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R&D Systems bmpr2 fc
Bmpr2 Fc, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bmpr2 fc/product/R&D Systems
Average 94 stars, based on 16 article reviews

bmpr2 fc - by Bioz Stars, 2026-05

94/100 stars

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GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

Techniques: Expressing, Transfection, Isolation, Western Blot, MANN-WHITNEY

GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

Techniques: Transfection, MANN-WHITNEY, Western Blot, Expressing

$Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).$

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

Techniques: Expressing, Injection

( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of BMPR2 in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of BMPR2 in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: RNA Sequencing, Knockdown, EdU Assay, Expressing, Infection

( a ) Progesterone (24 hr) enriched the genes of “MAPK pathways” and “Pathways in cancer” in BMRP2-knockdown PASMCs. ( b ) Progesterone (for 2 hr) activated the ERK pathway in BMPR2-knockdown PASMCs. ( c ) Venn graph, which brought into all the differentially-expressed genes with the fold change > 1.4, showed that MYC and EDN1 were upregulated by progesterone (24 hr) only under the condition of BMPR2 knockdown. ( d-e ) Progesterone upregulated the mRNA and protein of c-MYC and EDN1 in BMPR2-knockdown PASMCs. Abbreviation : ERK, extracellular regulated protein kinases; EDN1, endothelin1; MAPK, mitogen-activated protein kinase. **, P <0.01; ****, P <0.001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a ) Progesterone (24 hr) enriched the genes of “MAPK pathways” and “Pathways in cancer” in BMRP2-knockdown PASMCs. ( b ) Progesterone (for 2 hr) activated the ERK pathway in BMPR2-knockdown PASMCs. ( c ) Venn graph, which brought into all the differentially-expressed genes with the fold change > 1.4, showed that MYC and EDN1 were upregulated by progesterone (24 hr) only under the condition of BMPR2 knockdown. ( d-e ) Progesterone upregulated the mRNA and protein of c-MYC and EDN1 in BMPR2-knockdown PASMCs. Abbreviation : ERK, extracellular regulated protein kinases; EDN1, endothelin1; MAPK, mitogen-activated protein kinase. **, P <0.01; ****, P <0.001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Knockdown

( a ) Alarmar blue assay showed that PD0325901 and Bosentan (for 24 hr) reversed the pro-proliferative effects of progesterone in BMPR2-knockdown PASMCs . (b-c ) EdU assay showed that PD0325901 and Bosentan (for 24 hr) also reversed the effects of progesterone. ( d ) Inhibiting ERK pathway by PD0325901 reversed the upregulation of these proteins induced by progesterone in BMPR2-knockdown PASMCs. Abbreviation : ECE1, endothelin converting enzyme 1; PD, PD0325901; Bos, Bosentan. ns, non-significance; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a ) Alarmar blue assay showed that PD0325901 and Bosentan (for 24 hr) reversed the pro-proliferative effects of progesterone in BMPR2-knockdown PASMCs . (b-c ) EdU assay showed that PD0325901 and Bosentan (for 24 hr) also reversed the effects of progesterone. ( d ) Inhibiting ERK pathway by PD0325901 reversed the upregulation of these proteins induced by progesterone in BMPR2-knockdown PASMCs. Abbreviation : ECE1, endothelin converting enzyme 1; PD, PD0325901; Bos, Bosentan. ns, non-significance; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Knockdown, EdU Assay

( a-b ) Transfecting siPGR (200nM, 72 hr) effectively downregulated the mRNA and protein of PGR. ( c-e ) Knockdown of PGR reversed the pro-proliferative effects of progesterone (for 24 hr) in BMPR2-knockdown PASMCs. (c) used Alarmar blue assay, and (d-e) were EdU assay and its statistical graph. ( f ) Knockdown of PGR reversed the upregulation of these proteins induced by progesterone (for 2 hr) in BMPR2-knockdown PASMCs. Abbreviation: PGR, progesterone receptor. *, P <0.05; **, P <0.01; ***, P <0.001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a-b ) Transfecting siPGR (200nM, 72 hr) effectively downregulated the mRNA and protein of PGR. ( c-e ) Knockdown of PGR reversed the pro-proliferative effects of progesterone (for 24 hr) in BMPR2-knockdown PASMCs. (c) used Alarmar blue assay, and (d-e) were EdU assay and its statistical graph. ( f ) Knockdown of PGR reversed the upregulation of these proteins induced by progesterone (for 2 hr) in BMPR2-knockdown PASMCs. Abbreviation: PGR, progesterone receptor. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Knockdown, EdU Assay

( a-b ) Progesterone (for 2 hr) promoted the nuclear translocation of c-JUN in BMPR2– knockdown PASMCs, which could be reversed by knockdown of PGR. ( c ) Nucleus and cytoplasm ratio of mean fluorescence intensity of c-JUN. ( d ) Compared with RLP30 (the positive control) and IgG (the negative control), the %input of cJUN was over 4%, demonstrating its binding to the promoter region of EDN1. ( e ) JASPAR database predicted that AP-1 (the dimer of c-JUN and c-FOS) could combine onto the promoter region of EDN1. Abbreviation : ChIP, chromatin immunoprecipitation; AP-1, activator protein-1. **, P <0.01; ***, P <0.001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a-b ) Progesterone (for 2 hr) promoted the nuclear translocation of c-JUN in BMPR2– knockdown PASMCs, which could be reversed by knockdown of PGR. ( c ) Nucleus and cytoplasm ratio of mean fluorescence intensity of c-JUN. ( d ) Compared with RLP30 (the positive control) and IgG (the negative control), the %input of cJUN was over 4%, demonstrating its binding to the promoter region of EDN1. ( e ) JASPAR database predicted that AP-1 (the dimer of c-JUN and c-FOS) could combine onto the promoter region of EDN1. Abbreviation : ChIP, chromatin immunoprecipitation; AP-1, activator protein-1. **, P <0.01; ***, P <0.001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Translocation Assay, Knockdown, Fluorescence, Positive Control, Negative Control, Binding Assay, Chromatin Immunoprecipitation

( a ) Alarmar blue assay showed that progesterone (24 hr) upregulated the proliferation in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( b-c ) Transwell assay showed that progesterone upregulated the migration (8 hr) in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( d ) Progesterone (2 hr) induced the phosphorylation of ERK in iPSCs-VSMCs. ( e ) In comparison with normal iPSCs-VSMCs, EDN1 was significantly increased in PAH iPSCs-VSMCs that could be further upregulated by progesterone (24 hr). Abbreviation: iPSCs, induced pluripotent stem cells; VSMCs, vascular smooth muscle cells; HPAH, hereditary pulmonary artery hypertension; BMPR2 mut, BMRP2-mutation carrier; WT, wild type. ns, non-significance; *, P <0.05.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a ) Alarmar blue assay showed that progesterone (24 hr) upregulated the proliferation in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( b-c ) Transwell assay showed that progesterone upregulated the migration (8 hr) in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( d ) Progesterone (2 hr) induced the phosphorylation of ERK in iPSCs-VSMCs. ( e ) In comparison with normal iPSCs-VSMCs, EDN1 was significantly increased in PAH iPSCs-VSMCs that could be further upregulated by progesterone (24 hr). Abbreviation: iPSCs, induced pluripotent stem cells; VSMCs, vascular smooth muscle cells; HPAH, hereditary pulmonary artery hypertension; BMPR2 mut, BMRP2-mutation carrier; WT, wild type. ns, non-significance; *, P <0.05.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Transwell Assay, Migration, Phospho-proteomics, Comparison, Mutagenesis

( a ) Time flowchart of animal intervention and phenotype detection. ( b-c ) Right heart catheterization experiments showed the increase of RVSP in CKO and CKO+P group. (n=4 per group) ( d-e ) H&E stain showed the vascular remodeling of pulmonary arterioles (<50μM and 50-100μM) in CKO and CKO+P group and the statistical analyses (4 mice per group, 3 fields per mouse). ( f ) IF stain of vWF (green) and αSMA (red) showed the proliferation and hypertrophy of PASMCs in CKO and CKO+P group. Abbreviation: ♀, female; RVSP, right ventricle systolic pressure; CKO, SM22-cre BMPR2 flox +/- mice; P, progesterone; IF, immunofluorescence; vWF, von Willebrand factor; αSMA, smooth muscle actin. *, P <0.05; **, P <0.01; ****, P <0.0001.

Journal: bioRxiv

Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

doi: 10.1101/2023.04.21.537897

Figure Lengend Snippet: ( a ) Time flowchart of animal intervention and phenotype detection. ( b-c ) Right heart catheterization experiments showed the increase of RVSP in CKO and CKO+P group. (n=4 per group) ( d-e ) H&E stain showed the vascular remodeling of pulmonary arterioles (<50μM and 50-100μM) in CKO and CKO+P group and the statistical analyses (4 mice per group, 3 fields per mouse). ( f ) IF stain of vWF (green) and αSMA (red) showed the proliferation and hypertrophy of PASMCs in CKO and CKO+P group. Abbreviation: ♀, female; RVSP, right ventricle systolic pressure; CKO, SM22-cre BMPR2 flox +/- mice; P, progesterone; IF, immunofluorescence; vWF, von Willebrand factor; αSMA, smooth muscle actin. *, P <0.05; **, P <0.01; ****, P <0.0001.

Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

Techniques: Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Techniques: Expressing, Transfection, Control, Isolation, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Techniques: Transfection, Control, MANN-WHITNEY, Western Blot, Expressing

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Techniques: Expressing, Injection, Comparison

Loss of TLR3, p53, and BMPR2 converge on proliferative endothelial cells in the pulmonary arteriopathy of patients with PAH (A) Representative confocal microscopy optical sections of double immunofluorescence stainings (pseudo-colored) for proliferating cell nuclear antigen (PCNA)/Toll-like receptor 3 (TLR3), p53/von Willebrand Factor (vWF), and bone morphogenic protein receptor 2 (BMPR2)/vWF. For PCNA/TLR3, white arrows show intima cells in the PAH lesions with low TLR3 staining (red) that are positive for PCNA (green). In the plexiform lesions, asterisks indicate endothelial channels. For the p53/vWF staining, orange arrows show vWF + ECs with high nuclear p53 staining, whereas for the BMPR2/vWF staining, yellow arrows show vWF + ECs with high BMPR2 staining. (B and C) Quantification of mean fluorescence intensity in ECs/intima of representative images of pulmonary arteries from control and PAH patients. Data are shown as single values and mean ± SEM. ∗∗p < 0.01 (t test). (D) Representative confocal microscopy optical sections show proliferation in ECs and mesenchymal cells in pulmonary arteriopathy of patients with PAH. ECs are indicated by staining with Texas-Red-conjugated Lycopersicon esculentum lectin (tomato lectin, LEL, red pseudocolor), and mesenchymal cells are indicated by staining with fibroblast-specific protein 1 (FSP1, S100A4). Proliferation is indicated by expression of PCNA (gray pseudocolor). White arrows indicate LEL + PCNA + cells (proliferating ECs), and orange arrows indicate FSP-1 + PCNA + cells (proliferating mesenchymal lineage cells). The dotted squares indicate areas that are shown in greater detail in the images as indicated by thick yellow arrows. Nuclear staining with DAPI (blue). Scale bars: 50 μm, except for FSP-1/LEL/PCNA staining images that show more detail on far right, which have a scale bar of 20 μm. Images represent n = 3 control and n = 3 PAH patients.

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: Loss of TLR3, p53, and BMPR2 converge on proliferative endothelial cells in the pulmonary arteriopathy of patients with PAH (A) Representative confocal microscopy optical sections of double immunofluorescence stainings (pseudo-colored) for proliferating cell nuclear antigen (PCNA)/Toll-like receptor 3 (TLR3), p53/von Willebrand Factor (vWF), and bone morphogenic protein receptor 2 (BMPR2)/vWF. For PCNA/TLR3, white arrows show intima cells in the PAH lesions with low TLR3 staining (red) that are positive for PCNA (green). In the plexiform lesions, asterisks indicate endothelial channels. For the p53/vWF staining, orange arrows show vWF + ECs with high nuclear p53 staining, whereas for the BMPR2/vWF staining, yellow arrows show vWF + ECs with high BMPR2 staining. (B and C) Quantification of mean fluorescence intensity in ECs/intima of representative images of pulmonary arteries from control and PAH patients. Data are shown as single values and mean ± SEM. ∗∗p < 0.01 (t test). (D) Representative confocal microscopy optical sections show proliferation in ECs and mesenchymal cells in pulmonary arteriopathy of patients with PAH. ECs are indicated by staining with Texas-Red-conjugated Lycopersicon esculentum lectin (tomato lectin, LEL, red pseudocolor), and mesenchymal cells are indicated by staining with fibroblast-specific protein 1 (FSP1, S100A4). Proliferation is indicated by expression of PCNA (gray pseudocolor). White arrows indicate LEL + PCNA + cells (proliferating ECs), and orange arrows indicate FSP-1 + PCNA + cells (proliferating mesenchymal lineage cells). The dotted squares indicate areas that are shown in greater detail in the images as indicated by thick yellow arrows. Nuclear staining with DAPI (blue). Scale bars: 50 μm, except for FSP-1/LEL/PCNA staining images that show more detail on far right, which have a scale bar of 20 μm. Images represent n = 3 control and n = 3 PAH patients.

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Fluorescence, Control, Expressing

Reduced p53 protein level in PAECs from PAH patients and endothelial deletion of p53 exaggerates PH induced by SU5416 and chronic hypoxia (A and B) Representative western blot (A) and densitometric quantification (B) of p53 in pulmonary artery EC lines from n = 4 control and n = 4 PAH patients. β-actin is loading control. Data were normalized to the arithmetic mean of the control ECs. ∗∗p < 0.01 (t test). (C) Right ventricular systolic pressure (RVSP) and (D) Fulton index of male and female p53 wt Cdh5-Cre − , p53 wt Cdh5-Cre + , p53 flox Cdh5-Cre − , and p53 flox Cdh5-Cre + mice at day 21 after exposure to normoxia or cHx/Su. (E) Representative images of α-smooth muscle actin immunohistochemistry show increased pulmonary artery media wall thickness in p53 flox Cdh5-Cre + mice exposed to cHx/Su. Counterstaining with Meyer’s hematoxylin. (F) Media wall thickness (MWT) of pulmonary arteries. (G and H) Representative images (G) and quantification (H) of immunohistochemistry for proliferating cell nuclear antigen (PCNA). Arrows indicate PCNA + cells in PAs. Counterstaining with Meyer’s hematoxylin. (I) Representative optical sections obtained by confocal microscopy show reduced BMPR2 (green pseudocolor) and TLR3 (cyan pseudocolor) expression in Lycopersicon Esculentum lectin + (LEL, tomato lectin) endothelial cells (ECs) (red pseudocolor) in pulmonary arteries from cHx/Su-treated p53 flox Cdh5-Cre + mice. Arrows show ECs with BMPR2 and TLR3 staining, which is reduced in Cre + mice. Nuclear staining with DAPI. Overlay with differential interference contrast (DIC) demonstrates tissue structure. Images represent n = 3 per group. Scale bar: 25 μm. (C, D, F, H): ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA). All values are expressed as single data points and mean ± SEM. See also <xref ref-type=

Figure S1 . " width="100%" height="100%">

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: Reduced p53 protein level in PAECs from PAH patients and endothelial deletion of p53 exaggerates PH induced by SU5416 and chronic hypoxia (A and B) Representative western blot (A) and densitometric quantification (B) of p53 in pulmonary artery EC lines from n = 4 control and n = 4 PAH patients. β-actin is loading control. Data were normalized to the arithmetic mean of the control ECs. ∗∗p < 0.01 (t test). (C) Right ventricular systolic pressure (RVSP) and (D) Fulton index of male and female p53 wt Cdh5-Cre − , p53 wt Cdh5-Cre + , p53 flox Cdh5-Cre − , and p53 flox Cdh5-Cre + mice at day 21 after exposure to normoxia or cHx/Su. (E) Representative images of α-smooth muscle actin immunohistochemistry show increased pulmonary artery media wall thickness in p53 flox Cdh5-Cre + mice exposed to cHx/Su. Counterstaining with Meyer’s hematoxylin. (F) Media wall thickness (MWT) of pulmonary arteries. (G and H) Representative images (G) and quantification (H) of immunohistochemistry for proliferating cell nuclear antigen (PCNA). Arrows indicate PCNA + cells in PAs. Counterstaining with Meyer’s hematoxylin. (I) Representative optical sections obtained by confocal microscopy show reduced BMPR2 (green pseudocolor) and TLR3 (cyan pseudocolor) expression in Lycopersicon Esculentum lectin + (LEL, tomato lectin) endothelial cells (ECs) (red pseudocolor) in pulmonary arteries from cHx/Su-treated p53 flox Cdh5-Cre + mice. Arrows show ECs with BMPR2 and TLR3 staining, which is reduced in Cre + mice. Nuclear staining with DAPI. Overlay with differential interference contrast (DIC) demonstrates tissue structure. Images represent n = 3 per group. Scale bar: 25 μm. (C, D, F, H): ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA). All values are expressed as single data points and mean ± SEM. See also Figure S1 .

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Western Blot, Control, Immunohistochemistry, Confocal Microscopy, Expressing, Staining

Clonal expansion requires loss of p53, and p53 regulates TLR3 expression in rat lung CD117 + ECs and EC clones and human pulmonary artery ECs from PAH patients (A and B) qRT-PCR data show lower Tp53 and Tlr3 expression after four generations of clonal expansion in CD117 + lin − CD31 + rat lung ECs. (C) Clonal expansion was vastly reduced in CD117 + rat lung ECs when treated for 14 days with Nutlin 3a, which inhibits p53 protein degradation. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group. (D) qRT-PCR shows reduced mRNA expression of Tp53 , Tlr3 , and Id1 following siRNA targeting p53 versus control (scrambled, scrm) siRNA in rat lung CD117 + ECs. n = 5–6. (E and F) Representative images (scale bar: 250 μm, stitched images) and quantification of total network area and network length from 2D matrigel tube formation assay in CD117 + rat lung ECs after treatment with siRNA targeting p53 or scrambled (scrm) unspecific siRNA. n = 4 per group. (G and H) Representative western blots (G) and quantification of three independent experiments (H) show p53 protein accumulation following treatment with the MDM2 inhibitor Nutlin 3a. n = 9 per group total. (I) qRT-PCR shows increased expression of Tlr3 , Mdm2 , and Id1 following Nutlin 3a treatment. n = 6 per group. (J and K) Representative images (scale bar: 250 μm, stitched images) and quantification of total network area and network length in rat lung CD117 + fourth generation EC clones after treatment with Nutlin 3a or vehicle. n = 5 per group. (L and N) Representative western blots of p53, TLR3, BMPR2, phospho-(p)-IRF3, and p-Smad1/5/9 (L) and densitometric quantification of p53, TLR3, and BMPR2 from 2 independent experiments (n = 3 each) in PAECs from PAH patients following Nutlin 3a or vehicle treatment for 24 h. (O) qRT-PCR shows increased TLR3 mRNA expression following Nutlin 3a treatment in PAH PAECs. n = 5 per group. (P) Quantification of total network area and network length in PAH PAECs after treatment with Nutlin 3a or vehicle. n = 10 per group. Quantification data are n-fold of the arithmetic mean of veh. (Q) Clonal expansion was vastly reduced in PAECs following 14 days of treatment with Nutlin 3a. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group. All values are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (t test). See also <xref ref-type=

Figures S2–S9 . " width="100%" height="100%">

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: Clonal expansion requires loss of p53, and p53 regulates TLR3 expression in rat lung CD117 + ECs and EC clones and human pulmonary artery ECs from PAH patients (A and B) qRT-PCR data show lower Tp53 and Tlr3 expression after four generations of clonal expansion in CD117 + lin − CD31 + rat lung ECs. (C) Clonal expansion was vastly reduced in CD117 + rat lung ECs when treated for 14 days with Nutlin 3a, which inhibits p53 protein degradation. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group. (D) qRT-PCR shows reduced mRNA expression of Tp53 , Tlr3 , and Id1 following siRNA targeting p53 versus control (scrambled, scrm) siRNA in rat lung CD117 + ECs. n = 5–6. (E and F) Representative images (scale bar: 250 μm, stitched images) and quantification of total network area and network length from 2D matrigel tube formation assay in CD117 + rat lung ECs after treatment with siRNA targeting p53 or scrambled (scrm) unspecific siRNA. n = 4 per group. (G and H) Representative western blots (G) and quantification of three independent experiments (H) show p53 protein accumulation following treatment with the MDM2 inhibitor Nutlin 3a. n = 9 per group total. (I) qRT-PCR shows increased expression of Tlr3 , Mdm2 , and Id1 following Nutlin 3a treatment. n = 6 per group. (J and K) Representative images (scale bar: 250 μm, stitched images) and quantification of total network area and network length in rat lung CD117 + fourth generation EC clones after treatment with Nutlin 3a or vehicle. n = 5 per group. (L and N) Representative western blots of p53, TLR3, BMPR2, phospho-(p)-IRF3, and p-Smad1/5/9 (L) and densitometric quantification of p53, TLR3, and BMPR2 from 2 independent experiments (n = 3 each) in PAECs from PAH patients following Nutlin 3a or vehicle treatment for 24 h. (O) qRT-PCR shows increased TLR3 mRNA expression following Nutlin 3a treatment in PAH PAECs. n = 5 per group. (P) Quantification of total network area and network length in PAH PAECs after treatment with Nutlin 3a or vehicle. n = 10 per group. Quantification data are n-fold of the arithmetic mean of veh. (Q) Clonal expansion was vastly reduced in PAECs following 14 days of treatment with Nutlin 3a. Figures indicate the number of wells that yielded colonies from a single cell after 14 days. n = 4 per group. All values are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (t test). See also Figures S2–S9 .

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Expressing, Clone Assay, Quantitative RT-PCR, Control, Tube Formation Assay, Western Blot

Nutlin 3a treatment decreases pulmonary artery remodeling and PH in chronic hypoxia+CD117 + EC-clone-induced PH (A) RVSP and (B) RV hypertrophy index following Nutlin 3a or vehicle treatment from day 1–21 in cHx + EC clone PH male rats at day 21. (C and D) Representative images of von Willebrand factor (vWF) and α-smooth muscle actin (α-SMA) immunohistochemistry. Scale bar: 25 μm. (E) Echocardiographically measured pulmonary artery acceleration time (PAAT) following Nutlin 3a or vehicle treatment from day 1–21 in chronic hypoxia/EC clone PH male rats at day 21. (F) Quantification of open, partially occluded, and fully occluded pulmonary arteries in vehicle and Nutlin-3a treated chronic hypoxia/EC clone rats. (G and H) Media wall thickness (MWT) of small- (G) and medium-sized (H) pulmonary arteries. (I) Representative optical sections of p53 immunofluorescence staining (green pseudocolor) obtained by confocal microscopy show stronger p53 staining in pulmonary arteries, including nuclear accumulation in an EC (arrow) following Nutlin 3a treatment. Nuclear staining with DAPI. Scale bar: 25 μm. (J) Representative optical sections obtained by confocal microscopy show reduced BMPR2 (green pseudocolor) and TLR3 (cyan pseudocolor) expression in Lycopersicon Esculentum lectin + (LEL, tomato lectin) ECs (red pseudocolor) in pulmonary arteries from chronic hypoxia/EC clone + vehicle rats. Nutlin 3a treatment substantially increased BMPR2 and TLR3 staining. Yellow arrows show ECs with strong BMPR2 and TLR3 staining. Nuclear staining with DAPI. Overlay with differential interference contrast (DIC) demonstrates tissue structure. Scale bar: 25 μm. Images represent n = 3 per group. (K) Western blots from lung tissue homogenate of chronic hypoxia/EC clone rats treated with vehicle or Nutlin 3a demonstrates increased p53, phospho-(p)-Smad1-5-9, TLR3 and BMPR2 protein levels, and reduced PCNA protein expression. β-actin is loading control. (L) Densitometric quantification of the western blots in Κ versus β-actin. Data are normalized to the arithmetic mean of the vehicle controls. All values are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (t test, except for F: one-way ANOVA). See also <xref ref-type=

Figure S10 . " width="100%" height="100%">

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: Nutlin 3a treatment decreases pulmonary artery remodeling and PH in chronic hypoxia+CD117 + EC-clone-induced PH (A) RVSP and (B) RV hypertrophy index following Nutlin 3a or vehicle treatment from day 1–21 in cHx + EC clone PH male rats at day 21. (C and D) Representative images of von Willebrand factor (vWF) and α-smooth muscle actin (α-SMA) immunohistochemistry. Scale bar: 25 μm. (E) Echocardiographically measured pulmonary artery acceleration time (PAAT) following Nutlin 3a or vehicle treatment from day 1–21 in chronic hypoxia/EC clone PH male rats at day 21. (F) Quantification of open, partially occluded, and fully occluded pulmonary arteries in vehicle and Nutlin-3a treated chronic hypoxia/EC clone rats. (G and H) Media wall thickness (MWT) of small- (G) and medium-sized (H) pulmonary arteries. (I) Representative optical sections of p53 immunofluorescence staining (green pseudocolor) obtained by confocal microscopy show stronger p53 staining in pulmonary arteries, including nuclear accumulation in an EC (arrow) following Nutlin 3a treatment. Nuclear staining with DAPI. Scale bar: 25 μm. (J) Representative optical sections obtained by confocal microscopy show reduced BMPR2 (green pseudocolor) and TLR3 (cyan pseudocolor) expression in Lycopersicon Esculentum lectin + (LEL, tomato lectin) ECs (red pseudocolor) in pulmonary arteries from chronic hypoxia/EC clone + vehicle rats. Nutlin 3a treatment substantially increased BMPR2 and TLR3 staining. Yellow arrows show ECs with strong BMPR2 and TLR3 staining. Nuclear staining with DAPI. Overlay with differential interference contrast (DIC) demonstrates tissue structure. Scale bar: 25 μm. Images represent n = 3 per group. (K) Western blots from lung tissue homogenate of chronic hypoxia/EC clone rats treated with vehicle or Nutlin 3a demonstrates increased p53, phospho-(p)-Smad1-5-9, TLR3 and BMPR2 protein levels, and reduced PCNA protein expression. β-actin is loading control. (L) Densitometric quantification of the western blots in Κ versus β-actin. Data are normalized to the arithmetic mean of the vehicle controls. All values are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (t test, except for F: one-way ANOVA). See also Figure S10 .

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Confocal Microscopy, Expressing, Western Blot, Control

Increased TLR3 signaling promotes BMPR2 signaling in p53-deficient PAECs (A and B) Representative histograms of 5-bromodesoxyuridine (BrdU) pulsed (4h) and BrdU-antibody-stained (AF647) PAECs treated with adenoviruses expressing sh-scrm (control sh RNA), sh-TP53, and sh-TP53 combined with 25 μg/mL Poly(I:C). (B) Quantification from 2 different cell lines (total n = 6 each). Data indicate increased DNA synthesis in sh-TP53 cells, which is reduced by Poly(I:C). Data are shown after normalization to the arithmetic mean of the sh-scrm group. (C and D) Representative phase contrast images (scale bar: 500 μm, stitched images, C) and quantification of total network area and network length (D) in rat lung CD117 + fourth generation EC clones after treatment with Nutlin 3a or vehicle. Two different cell lines (total n = 12 each). Data are shown after normalization to the arithmetic mean of the sh-scrm group. (E and F) Western blots (E) and densitometric quantification (F) of TLR3, p-IRF3, BMPR2, p-Smad1/5/9, and Smad1 in PAECs treated with sh-scrm, sh-TP53, and sh-TP53 + Poly(I:C). The results have been expressed as a ratio of the loading control β-actin and normalized to the arithmetic mean of the sh-scrm group. (G) qRT-PCR quantification of steady-state mRNA levels of TP53 , TLR3 , CXCL10 , BMP2 , and BMP4 . The results are expressed as a ratio of the housekeeping gene and normalized to the arithmetic mean of the sh-scrm group. The data are shown as single data points and mean ± SEM. Data are shown as single data points and mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA).

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: Increased TLR3 signaling promotes BMPR2 signaling in p53-deficient PAECs (A and B) Representative histograms of 5-bromodesoxyuridine (BrdU) pulsed (4h) and BrdU-antibody-stained (AF647) PAECs treated with adenoviruses expressing sh-scrm (control sh RNA), sh-TP53, and sh-TP53 combined with 25 μg/mL Poly(I:C). (B) Quantification from 2 different cell lines (total n = 6 each). Data indicate increased DNA synthesis in sh-TP53 cells, which is reduced by Poly(I:C). Data are shown after normalization to the arithmetic mean of the sh-scrm group. (C and D) Representative phase contrast images (scale bar: 500 μm, stitched images, C) and quantification of total network area and network length (D) in rat lung CD117 + fourth generation EC clones after treatment with Nutlin 3a or vehicle. Two different cell lines (total n = 12 each). Data are shown after normalization to the arithmetic mean of the sh-scrm group. (E and F) Western blots (E) and densitometric quantification (F) of TLR3, p-IRF3, BMPR2, p-Smad1/5/9, and Smad1 in PAECs treated with sh-scrm, sh-TP53, and sh-TP53 + Poly(I:C). The results have been expressed as a ratio of the loading control β-actin and normalized to the arithmetic mean of the sh-scrm group. (G) qRT-PCR quantification of steady-state mRNA levels of TP53 , TLR3 , CXCL10 , BMP2 , and BMP4 . The results are expressed as a ratio of the housekeeping gene and normalized to the arithmetic mean of the sh-scrm group. The data are shown as single data points and mean ± SEM. Data are shown as single data points and mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA).

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Staining, Expressing, Control, DNA Synthesis, Clone Assay, Western Blot, Quantitative RT-PCR

TLR3/IRF3 signaling maintains lung endothelial BMPR2 expression in vitro (A and B) Representative western blots (A) and densitometric quantification (B) of TLR3, BMPR2, and p-Smad1/5/9 in rat lung CD117 + ECs treated with sh-scrm (control-shRNA) and sh-Tlr3 (shRNA targeting Tlr3). The results have been expressed as a ratio of the loading control β-actin and normalized to the arithmetic mean of the sh-scrm group. The data are expressed as mean ± SEM. (C) qRT-PCR shows reduced Tlr3 and Bmpr2 steady-state mRNA level after sh-Tlr3 treatment of rat lung CD117 + ECs. The results have been expressed as a ratio of the housekeeping gene and normalized to the arithmetic mean of the sh-scrm group. The data are shown as mean ± SEM. (D and E) Representative immunoblot (D) and densitometric quantification (E) confirm the efficiency of the TLR3 CRISPR/cas9 gene editing in human PAECs. (F and G) Representative differential interference contrast (DIC) images (stitched by software) (F) and quantification of total network area and network length (G) in control PAECs that underwent gene editing using CRISPR/Cas9 technology with sgRNA targeting TLR3 or unspecific (scrm) sgRNA. The results have been normalized to the arithmetic mean of the scrm sgRNA group. Scale bar: 500μm. (H and I) Representative western blots (H) and densitometric quantification (I) indicate reduced BMPR2 expression in IRF3 siRNA-treated PAECs. The results have been normalized to the arithmetic mean of the si scrm group. (J) Diagram showing the location targeted by the primers pairs for qPCR amplification after chromatin immunoprecipitation (ChIP) for the predicted IRF3 binding sites (1A-C and 2A-C) upstream of the human BMPR2 TSS. The Eukaryotic Promoter Database, which was used for consensus site prediction, provided two version of the human BMPR2 promoter, BMPR2_1 and BMPR2_2 , with different starting positions on the chromosome. (K) ChIP qPCR indicates IRF3 chromatin immunoprecipitation and amplification of predicted consensus sites with the specific IRF3 antibody, but not with IgG control antibody, suggesting specific binding of IRF3 at the predicted consensus sites. In addition, Poly(I:C) treatment (25 μg/mL, 24h) promoted IRF3 binding (=amplification) at all consensus sites upstream of BMPR2 TSS. Because of the number of potential consensus sites, data from all consensus sites were integrated for analysis using two-way ANOVA, demonstrating a significant increase in IRF3 binding with Poly(I:C) treatment [p = 0.0049 Poly(I:C) versus control]. Data are shown as single data points and mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (t test). See also <xref ref-type=

Figures S11–S13 . " width="100%" height="100%">

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet: TLR3/IRF3 signaling maintains lung endothelial BMPR2 expression in vitro (A and B) Representative western blots (A) and densitometric quantification (B) of TLR3, BMPR2, and p-Smad1/5/9 in rat lung CD117 + ECs treated with sh-scrm (control-shRNA) and sh-Tlr3 (shRNA targeting Tlr3). The results have been expressed as a ratio of the loading control β-actin and normalized to the arithmetic mean of the sh-scrm group. The data are expressed as mean ± SEM. (C) qRT-PCR shows reduced Tlr3 and Bmpr2 steady-state mRNA level after sh-Tlr3 treatment of rat lung CD117 + ECs. The results have been expressed as a ratio of the housekeeping gene and normalized to the arithmetic mean of the sh-scrm group. The data are shown as mean ± SEM. (D and E) Representative immunoblot (D) and densitometric quantification (E) confirm the efficiency of the TLR3 CRISPR/cas9 gene editing in human PAECs. (F and G) Representative differential interference contrast (DIC) images (stitched by software) (F) and quantification of total network area and network length (G) in control PAECs that underwent gene editing using CRISPR/Cas9 technology with sgRNA targeting TLR3 or unspecific (scrm) sgRNA. The results have been normalized to the arithmetic mean of the scrm sgRNA group. Scale bar: 500μm. (H and I) Representative western blots (H) and densitometric quantification (I) indicate reduced BMPR2 expression in IRF3 siRNA-treated PAECs. The results have been normalized to the arithmetic mean of the si scrm group. (J) Diagram showing the location targeted by the primers pairs for qPCR amplification after chromatin immunoprecipitation (ChIP) for the predicted IRF3 binding sites (1A-C and 2A-C) upstream of the human BMPR2 TSS. The Eukaryotic Promoter Database, which was used for consensus site prediction, provided two version of the human BMPR2 promoter, BMPR2_1 and BMPR2_2 , with different starting positions on the chromosome. (K) ChIP qPCR indicates IRF3 chromatin immunoprecipitation and amplification of predicted consensus sites with the specific IRF3 antibody, but not with IgG control antibody, suggesting specific binding of IRF3 at the predicted consensus sites. In addition, Poly(I:C) treatment (25 μg/mL, 24h) promoted IRF3 binding (=amplification) at all consensus sites upstream of BMPR2 TSS. Because of the number of potential consensus sites, data from all consensus sites were integrated for analysis using two-way ANOVA, demonstrating a significant increase in IRF3 binding with Poly(I:C) treatment [p = 0.0049 Poly(I:C) versus control]. Data are shown as single data points and mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (t test). See also Figures S11–S13 .

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Expressing, In Vitro, Western Blot, Control, shRNA, Quantitative RT-PCR, CRISPR, Software, Amplification, Chromatin Immunoprecipitation, Binding Assay, ChIP-qPCR

Journal: iScience

Article Title: A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

doi: 10.1016/j.isci.2023.105935

Figure Lengend Snippet:

Article Snippet: Human BMPR2 , Qiagen , QT02317196.

Techniques: Clone Assay, Isolation, Recombinant, Plasmid Preparation, Membrane, Microscopy, Electron Microscopy, Amplification, Reverse Transcription, SYBR Green Assay, Software

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